This project examines the role of protein acetylation in mediating nutrient-induced mitochondrial dysfunction. Lysine acetylation (AcK) has emerged as an important reversible post-translational modification. The biochemical environment of the mitochondrial compartment is particularly favorable for acetylation and recent studies have identified multiple AcK sites on a large number of mitochondrial proteins, including several metabolic enzymes and proteins involved in oxidative phosphorylation. Increased mitochondrial AcK levels correlate with metabolic dysfunction in the context of aging and obesity. A growing body of evidence suggests that acetylation of specific lysine residues on mitochondrial proteins impairs metabolic regulation leading to glucose intolerance, and compromises skeletal muscle performance during exercise. The protein responsible for deacetylation within the mitochondria, sirtuin 3 (SIRT3), has been identified. However the molecular events that promote mitochondrial AcK remain poorly understood. Acetyl-coenzyme A (CoA), a metabolic intermediate of glucose and fat catabolism, is the proposed substrate required for reversible acetylation. In vitro studies suggest mitochondrial AcK can occur non-enzymatically as a result of elevated mitochondrial acetyl-CoA production and/or accumulation. In addition, recent studies of the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT) underscore an important link between elevated acetyl-CoA levels, mitochondrial AcK and metabolic regulation. CrAT converts acetyl-CoA to its membrane permeate carnitine conjugate, acetylcarnitine. The CrAT reaction permits the mitochondrial efflux of excess acetyl groups and thereby has the potential to influence AcK events that are driven by mass action. This prediction is supported by preliminary studies from the laboratory showing that CrAT deficiency in mice exacerbates mitochondrial AcK in response to nutrient excess. Furthermore, CrAT ablation in the skeletal muscle results in metabolic dysfunction and exercise intolerance. The objective of this proposal is to determine if lysine acetylation of mitochondrial proteins contributes to the metabolic consequences of CrAT deficiency. The proposed studies will focus upon the molecular and physiological characterization of CrAT deficient cells and mice. First, we will test the hypothesis that stimuli that decrease mitochondrial acetylation can restore metabolic function in CrAT deficient myocytes. Second, we will use mice with a muscle-specific deletion of CrAT to determine whether normalization of mitochondrial AcK status restores glucose and/or exercise tolerance in the absence muscle CrAT. The results from these studies will expand our understanding of the functional relevance of mitochondrial AcK and could lead to the identification of therapeutic targets for treating age- and obesity- related skeletal muscle metabolic dysfunction and exercise intolerance.